Bicinchoninic acid protein assay kit

The iSCAPs were seeded in 24-well plates and infected with indicated multiplicty of infection (MOI) values of AdGFP, AdBMP9, or AdsiBMP9. Polybrene (10 μg/mL) was used to enhance transduction efficiency for adenoviral infection. At 48 h after the infection, GFP signal or red fluorescent protein (RFP) signal of the infected iSCAPs was assessed under a fluorescence microscope (Carl Zeiss Microimaging GmbH, Gottingen, Germany). The infection efficiency was indicated by the GFP or RFP expression proportion of the cells. Alkaline phosphatase (ALP) histochemical staining was carried out by using the NBT/BCIP staining kit (Beyotime-Bio, China), and ALP activity was assessed quantitatively with the ALP assay kit (Beyotime-Bio, China) on day 5. Each assay condition was performed in triplicate, and the results were repeated in at least three independent experiments. ALP activity was normalized by total cellular protein concentrations determined by the bicinchoninic acid Protein Assay Kit (Beyotime-Bio, China).

Proteins were extracted using radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Inc.) and the protein content was determined using the Bicinchoninic Acid Protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of the protein (50 µg/lane) from lysates of PANC-1 cells were subjected to SDS-PAGE (10% gels) and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 60 min at room temperature, followed by incubation with primary antibodies against HSPA2 (cat. no. ab108416; 1:500; Abcam, Cambridge, MA, USA) and GAPDH (cat. no. AF0006; 1:1,000; Beyotime Institute of Biotechnology) overnight at 4°C. Membranes were then washed with 0.1% Tween-20 in PBS (PBST) three times at room temperature. Then, they were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (cat. no. sc-2004; 1:3,000; Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) at room temperature for 1 h. The proteins of interest were detected by the enhanced chemiluminescence detection system (Sea Biotech, Shanghai, China). Finally, the intensity of protein bands was detected using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). GAPDH served as the loading control.

The mouse spinal cord tissues (L4–6) were harvested and homogenized using RIPA buffer (Beyotime, P0013B) supplemented with 1× protease inhibitor cocktail (Sigma‐Aldrich; P8304), phosphatase inhibitor cocktail II and III (Sigma‐Aldrich; P5726). The supernatant was collected by centrifugation at 12,000g for 10 min, and the protein concentration was detected using a bicinchoninic acid protein assay kit (Beyotime, P0012S). An aliquot of 50 μg protein from each sample was separated using SDS‐PAGE and transferred to a PVDF membrane, then blocked with 5% nonfat milk in TBST (pH 7.4). Thereafter, the membranes were incubated with primary antibodies including arginase I (1:1000; CST; #93668), argininosuccinate synthetase (1:1000; abcam; ab17095), argininosuccinatelyase (1:1000; abcam; ab97370) and actin (1:1000; ABclonal; AC026). After incubation with the appropriate horseradish peroxidase (HRP) conjugated secondary antibodies (IgG, against rabbit, 1:1000; ABclonal; AS014), the immune complexes were visualized using the SuperSignal West Pico Substrate (34,077, Pierce). The digital images were quantified using densitometric measurements by Quantity‐One software (Bio‐Rad).

The total protein was extracted in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China), and the concentration of protein was quantified using a bicinchoninic acid protein assay kit (Beyotime). Equal amounts (20 µg/lane) of protein were fractionated using SDS-PAGE and transferred to polyvinylidene difluoride membranes. These membranes were then incubated with anti-Bax, anti-Caspase-3, anti-Cleaved Caspase-3, anti-Bcl-2, anti-cyclin B1, anti-cyclin D1, anti-P21, anti-CDC21, anti-PI3K, anti-p-mTOR, anti-mTOR, anti-p-EKR1/2, anti-EKR1/2, anti-pAKT, anti-AKT, and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA). Next the membranes were incubated with peroxidase-conjugated secondary antibodies. The Western blot bands were visualized using electrochemi-luminescence kits in accordance with the manufacturer’s instructions (Abcam, Cambridge, UK). GAPDH was used for normalization of protein loading.